Mechanistic Principles Behind Molecular Mechanism of Rifampicin Resistance in Mutant RNA Polymerase Beta Subunit of Mycobacterium tuberculosis.

Evolution of drug-resistant Mycobacterium strains threatens the TB treatment and control programs globally. Rifampicin (RIF) is an important first line antitubercular drug. Resistance to Rifampicin is caused mainly by mutations in its target RNA polymerase beta subunit protein (RpoB). RpoB contains a Rifampicin resistance determining region (RRDR) and has several potent sites for mutations. In this study, we have investigated mutations of a single site (H451) to eight different amino acids, involved in RIF resistance. Long-term molecular dynamics simulations were performed on wild type (WT) and mutant protein structures and various structural analysis were carried out to elucidate the dynamic behavior of WT and mutant forms. Essential dynamics uncovered the difference in conformational flexibility and collective modes of motions between WT and mutants. MMPBSA calculations and interaction pattern analysis revealed the binding site relocation in some mutants. This study presents an exhaustive analysis of RIF binding to the WT and mutant RpoB and clearly highlights structural mechanism for differences in stable binding of Rifampicin with WT than the mutant targets. J. Cell. Biochem. 118: 4594-4606, 2017. © 2017 Wiley Periodicals, Inc.

Cell Transdifferentiation and Reprogramming in Disease Modeling: Insights into the Neuronal and Cardiac Disease Models and Current Translational Strategies.

Cell transdifferentiation and reprogramming approaches in recent times have enabled the manipulation of cell fate by enrolling exogenous/artificial controls. The chemical/small molecule and regulatory components of transcription machinery serve as potential tools to execute cell transdifferentiation and have thereby uncovered new avenues for disease modeling and drug discovery. At the advanced stage, one can believe these methods can pave the way to develop efficient and sensitive gene therapy and regenerative medicine approaches. As we are beginning to learn about the utility of cell transdifferentiation and reprogramming, speculations about its applications in translational therapeutics are being largely anticipated. Although clinicians and researchers are endeavoring to scale these processes, we lack a comprehensive understanding of their mechanism(s), and the promises these offer for targeted and personalized therapeutics are scarce. In the present report, we endeavored to provide a detailed review of the original concept, methods and modalities enrolled in the field of cellular transdifferentiation and reprogramming. A special focus is given to the neuronal and cardiac systems/diseases towards scaling their utility in disease modeling and drug discovery.

Molecular mechanism of acetoacetyl-CoA enhanced kinetics for increased bioplastic production from Cupriavidus necator 428.

Polyhydroxyalkanoates are gaining importance due to their biodegradable nature and close analogy to plastics. Polyhydroxybutyrate (PHB) is the most widely used bioplastic from polyalkanoate family, which is produced by a legion of bacterial species via phbCAB operon encoding ß-ketothiolase (PhaA), NADPH-dependent acetoacetyl-coenzyme A (acetoacetyl-CoA) reductase (PhaB) and polyhydroxyalkanoate synthase (PhaC). Augmentation in the activity of these enzymes is promising for increased PHB production which is achieved by enzyme engineering strategies including non-structural and structural approaches. Our study is deployed on directed evolution-based experimentally reported mutants of PhaB enzyme with increased efficiency due to impact on critical structural factors. We have analyzed and compared the native PhaB with two of its variants Q47L and T173S in complex with their cofactor i.e. NADPH as well as the substrate i.e. acetoacetyl-CoA, via long range molecular dynamics simulations. Interaction profile, MMPBSA, essential dynamics, and free energy landscape analysis revealed that the enzyme efficiency is critically affected by cofactor interactions. It was also observed that mutants have higher equilibrium constant with lesser but optimal affinity for substrate and cofactor than the wild type, which might be the reason for increased efficiency of the mutants via enhanced substrate and cofactor exchange rate. Our study provides insights into the cofactor and substrate binding affinities to PhaB enzyme at atomistic level, which will facilitate designing of highly efficient PhaB enzymes for increased PHB production. Communicated by Ramaswamy H. Sarma.